Somatic epigenetic alterations in DNA methylation are tightly linked to development, cell differentiation and neoplastic transformation. For instance, hypermethylation of CpG islands in promoter regions has been increasingly associated with transcriptional inactivation of tumor suppressor genes in carcinogenesis. Although techniques to determine the degree of methylation in specific DNA segments or in total DNA have been available, there are few techniques to efficiently scan and identify changes in methylation in the entire genome. We have developed a method called Methylation Sensitive-Amplified Fragment Length Polymorphism (MS-AFLP). This PCR-based unbiased DNA fingerprinting technique permits the identification of the cleavage sites that exhibit DNA methylation alterations and subsequently allows the isolation of DNA fragments with these sites at their ends. Hyper/hypomethylation can easily be differentiated by the decrease/increase of band intensity, respectively. MS-AFLP requires low amounts of template DNA and electrophoresis of multiple samples in parallel enables easy identification of consistent common differences. Notl-Msel MS-AFLP experiments using matched normal/tumor DNA have shown highly reproducible differences in banding patterns some of which were specifically linked with the tumor phenotype. Sequencing some of these bands has identified multiple numbers of homeotic genes and the genes involved in the regulation of homeotic gene expression. These results demonstrate the potential of MS-AFLP in identifying epigenetic alterations associated with cell differentiation and cancer. We will further develop this powerful MS-AFLP method by transforming the gel electrophoresis-based fingerprinting technique into a DNA microarray-based hybridization technique for general use of methylation alteration analysis of several biological problems. In the R21 phase, we will construct a pilot DNA microarray panel, examine the feasibility and sensitivity of several hybridization-based MS-AFLP and non-PCR methods using the pilot DNA microarray, and determine the best method(s) for further development. In the R33 phase, we will search for the prostate and breast cancer-specific DNA methylation alterations, analyze the gene expression, re-examine some of the identified alterations in DNA methylation and gene expression by the sodium bisulfite modification method and multiplex RT -PCR. We will also construct a cancer-specific DNA microarray for the clinical detection of DNA methylation alterations.